During the development of the vertebrate central nervous system (CNS), hundreds of distinct neuronal types are generated, establishing a diversity that is essential for the formation of neuronal circuits. The degeneration of specific classes of CNS neurons is the hallmark of many neurological disorders, a realization that has prompted interest in defining proliferative cell populations that could serve as replenishable sources of neurons for the treatment of neurodegenerative diseases. Studies by a number of different groups have provided evidence that murine embryonic stem cells can be directed along specific pathways of neuronal differentiation in a systematic manner (Bain et al., 1995; Kawasaki et al., 2000; Munoz-Sanjuan 35 al., 2002; Tropepe et al., 2001; Uchida et al., 2000), raising the possibility that such stem cell-derived neurons could have clinical utility (Gage, 2000).
Generation of neuronal diversity during the vertebrate CNS development involves multiple steps, beginning with neural induction and patterning of the neural plate into broad anteroposterior domains: the prospective forebrain, midbrain, hindbrain and spinal cord (Muhr et al., 1999) Subsequently, neuroepithlial cells within each domain are patterned along dorso-ventral (DV) and antero-posterior (AP) axes to establish the principal fate map for future neurons.
Spinal motor neurons represent the class of CNS neuron that is perhaps best understood, both in the context of their mature function, and their developmental origins (Hollyday, 1980; Jessell, 2000; Lee and Pfaff, 2001). The DV patterning of the spinal cord leads to specification of ˜15 distinct classes of neurons, including the ventral MNs. Generic MN identity is established by the joint actions of two extrinsic signals: a long range gradient of Sonic hedgehog (Shh) activity provided by the notochord and floor plate, and a more diffuse influence of retinoid signals provided by the paraxial mesoderm (Briscoe and Ericson, 2001; Briscoe et al., 2000; Novitch et al., 2003).
Cells in the developing neural tube interpret these two signals by expressing a set of homeodomain proteins that define five principal progenitor domains within the ventral half of the spinal cord (Jessell, 2000; Lee and Pfaff, 2001). One of these—the pMN domain—which is marked by the expression of homeodomain transcription factor Nkx6.1 and basic helix-loop-helix (bHLH) transcription factor Olig2 is the sole source of motor neuron progenitors (Briscoe et al., 2000; Mizuguchi et al., 2001; Novitch et al., 2001; Zhou and Anderson, 2002). Expression of Olig2 within MN progenitors leads to the induction of pro-neural gene Neurogenin 2 (Ngn2) that governs cell cycle exit, acquisition of pan-neuronal identity, and induction of a set of transcription factors (Hb9, Lhx3 and Isl1) transiently expressed in all nascent spinal MNs, thus specifying generic motor neuron identity (Briscoe and Ericson, 2001).
With this information about the normal pathway of motor neuron generation, it has become possible to examine whether embryonic stem (ES) cells can respond to the same extrinsic signals to generation post-mitotic motor neurons through the same molecular pathway. Studies over the past few years have revealed that mouse ES cells can indeed generate spinal motor neurons at high efficiency, and that the pathway of motor neuron (MN) generation from ES cells recapitulates the steps of motor neuron generation in vivo. (Renoncourt et al., 1998; Wichterle et al., 200). ES cell-derived MNs in vitro acquire electrophysiological properties that resemble their embryo-derived counterparts, they develop appropriate ionic currents in response to neurotransmitters, they can receive synaptic inputs and fire repetitively at rates sufficient for functional muscle contract and they form functional synapses with cultured muscle cells (Miles et al., 2004).
Moreover, ES cell-derived MNs can repopulate the embryonic and adult spinal cord in vivo (Wichterle et al., 2002). In an embryonic environment ES cell MNs can extend axons into the periphery and form synapses with muscle targets (Wichterle et al., 2002). In an embryonic environment ES cell MNs can extend axons into the periphery and form synapses with muscle targets (Wichterle et al., 2002). In an adult environment, in which MNs degenerate, some limited axon extension out of the spinal cord is observed under pharmacological conditions that promote axonal regeneration (Harper et al., 2004). Together, these studies have indicated the feasibility of applying insights into normal developmental signaling cascades, in particular the control of extracellular inductive signals, to direct the differentiation of ES cells into spinal MNs. The potential for ES cell-derived MNs to innervate target muscle cells thus opens the way for a systematic evaluation of the use of such neuron to restore motor function, initially in mammalian models of spinal cord injury and motor neuron degenerative diseases.
There are, however, challenges in the basic study of cell differentiation into MNs which remain unaddressed.
All the studies performed on ES cell differentiation into MNs have assessed simply a set of generic motor neuron properties (Wichterle et al., 2002). Yet in the intact spinal cord, there are approximately one hundred different classes of MNs, each acquiring subtype specializations that are critical for the effective innervations of their cognate muscle and neuronal targets. Given the extensive evidence for MN specialization in situ, it remains unclear whether ES cells-derived MNs are capable of acquiring these highly specialized MN subtype characters. This is in part due to limited understanding of developmental processes and transcriptional programs controlling neuronal subtype diversification and in part due to technically demanding analysis of neuronal migratory and axon pathfinding properties by transplantation into the developing mammalian embryo.
At this point, no systematic effort has been made to regulate MN subtype identity in a developmentally sensible manner. Moreover, due to the state of knowledge of brachial Motor Neurons (bMNs) in comparison to cervical spinal motor neurons, methods for directed differentiation of ES cells into brachial spinal motor neuron and other more caudal motor neurons in vitro which can produce MNs that can acquire similarly complex and specialized subtype phenotypes as MN generated in the same section of the spinal cord in vivo needs to be investigated.